Amyloid formation in denatured single-mutant lysozymes where residual structures are modulated

Reduced hen lysozyme has a residual structure involving long-range interaction. It has been demonstrated that a single mutation (A9G, W62G, W111G, or W123G) in the residual structure differently modulates the long-range interactions of reduced lysozyme. To examine whether such variations in the residual structure affect amyloid formation, reduced and alkylated mutant lysozymes were incubated under the amyloid-fibrillation condition. From the analyses of CD spectra and thioflavine T fluorescences, it was suggested that variation in residual structure led to different amyloid formation. Interestingly, the extent of amyloid formation did not always correlate with the extent to which the residual structure was maintained, resulting in the involvement of a hydrophobic cluster normally contained in W111 in the reduced lysozyme.     

活性污泥微生物解壳聚糖松江菌中菌胶团形成相关大型基因簇的鉴定和分析

【背景】活性污泥法已广泛应用于城市污水和工业废水的处理,微生物菌胶团的形成在污泥通过重力沉淀实现泥水分离和污泥回用的过程中起着重要作用。从西安北石桥污水处理厂活性污泥中分离到一株菌胶团形成菌XHY-A6,经鉴定为解壳聚糖松江菌(Mitsuariachitosanitabida)。【目的】旨在揭示该株解壳聚糖松江菌菌胶团形成相关的基因及其菌胶团形成机制。【方法】结合分子遗传学,包括转座子插入突变技术和遗传互补分析以及基因组学方法分析与菌胶团形成相关的基因和基因簇。【结果】通过转座子插入突变技术获得了两株菌胶团形成缺陷的突变株,转座子插入位点在糖基转移酶(称为gt3)和多糖链长决定蛋白(wzz)基因内,且这两个基因位于一个与菌胶团形成相关的大型基因簇内,该基因簇内还包括与胞外多糖生物合成和分泌相关的基因、epsB2-prsK-psrR-prsT基因以及一个编码PEP-CTERM蛋白A的基因,遗传互补分析证明gt3基因、wzz基因及其下游wzc基因在菌胶团形成过程中是必需的。【结论】松江菌中菌胶团形成和调控机制极可能与活性污泥优势菌动胶菌(Zoogloea)非常相似,即由胞外多糖和PEP-CTERM家族胞外蛋白质共同介导。从武汉二郎庙、汤逊湖和深圳南山污水处理厂活性污泥中分离纯化出松江菌,这些松江菌属细菌可以用于富含几丁质和壳聚糖的市政污水和虾蟹类食品加工废水的净化和资源化利用。     

Diffusivity of oxygen in aerobic granules

This work for the first time estimated apparent oxygen diffusivity (D(app)) of two types of aerobic granules, acetate-fed and phenol-fed, by probing the dissolved oxygen (DO) level at the granule center with a sudden change in the DO of the bulk liquid. With a high enough flow velocity across the granule to minimize the effects of external mass transfer resistance, the diffusivity coefficients of the two types of granules were estimated with reference to a one-dimensional diffusion model. The carbon source has a considerable effect on the granule diameter (d) and the oxygen diffusivity. The diffusivity coefficients were noted 1.24-2.28 x 10(-9) m2/s of 1.28-2.50 mm acetate-fed granules, and 2.50-7.65 x 10(-10) m2/s of 0.42-0.78 mm phenol-fed granules. Oxygen diffusivity declined with decreasing granule diameter, in particular, the diffusivity of acetate-fed granules is proportional to the size, whereas the diffusivity of phenol-fed granules is proportional to the square of granule diameter. The existence of large pores in granule, evidenced by FISH-CLSM imaging, was proposed to correspond to the noted size-dependent oxygen diffusivity. The phenol-fed granules exhibited a higher excellular polymer (ECP) content than the acetate-fed granules, hence yielding a lower oxygen diffusivity.     

Germ cell cluster formation and cell death are alternatives in caste-specific differentiation of the larval honey bee ovary

Summary

Caste-specific differentiation of the female honey bee gonad takes place in the fifth larval instar. In queen larvae most ovarioles exhibit almost simultaneous formation of numerous germ cell clusters within the first 20 h after the last larval molt. Ultrastructurally distinctive fusomal cytoplasm connects these cystocytes. Germ cell differentiation is accompanied by morphological changes in somatic components of the ovarioles, the follicle and the terminal filament cells. Subsequently, queen ovarioles elongate and differentiate basal stalks that coalesce in a basal calyx. A second round of mitotic activity was found to occur in the late prepupal and early pupal queen ovary. This round may elevate germ cell numbers composing each cluster to levels observed in follicles of adult honey bee queens. In contrast, germ cell cluster formation does not occur in most of the 120–160 ovarioles of the larval worker ovary, but instead many cells in such ovarioles show signs of impending degeneration, such as large autophagic bodies. DNA extracted from worker ovaries did not reveal nucleosomal laddering, and ultrastructurally, chromatin in germ cell nuclei appeared intact. In the 4–7 surviving ovarioles of the small worker ovary, germ cell clusters were found with ultrastructural characteristics identical to those in queen ovarioles. The temporal window during which divergence in developmental pathways of the larval ovaries initiates shortly after the last larval molt coincides with caste-specific differences in juvenile hormone titer which have long been considered critical to caste-specific morphogenesis.     

Charge-mediated Fab-Fc interactions in an IgG1 antibody induce reversible self-association,cluster formation,and elevated viscosity

Concentration-dependent reversible self-association (RSA) of monoclonal antibodies (mAbs) poses a challenge to their pharmaceutical development as viable candidates for subcutaneous delivery. While the role of the antigen-binding fragment (Fab) in initiating RSA is well-established, little evidence supports the involvement of the crystallizable fragment (Fc). In this report, a variety of biophysical tools, including hydrogen exchange mass spectrometry, are used to elucidate the protein interface of such non-covalent protein-protein interactions. Using dynamic and static light scattering combined with viscosity measurements, we find that an IgG1 mAb (mAb-J) undergoes RSA primarily through electrostatic interactions and forms a monomer-dimer-tetramer equilibrium. We provide the first direct experimental mapping of the interface formed between the Fab and Fc domains of an antibody at high protein concentrations. Charge distribution heterogeneity between the positively charged interface spanning complementarity-determining regions CDR3H and CDR2L in the Fab and a negatively charged region in C_H3/Fc domain mediates the RSA of mAb-J. When arginine and NaCl are added, they disrupt RSA of mAb-J and decrease the solution viscosity. Fab-Fc domain interactions between mAb monomers may promote the formation of large transient antibody complexes that ultimately cause increases in solution viscosity. Our findings illustrate how limited specific arrangements of amino-acid residues can cause mAbs to undergo RSA at high protein concentrations and how conserved regions in the Fc portion of the antibody can also play an important role in initiating weak and transient protein-protein interactions.     

The hydrodynamic and conformational properties of denatured proteins in dilute solutions

Published data on the characterization of unfolded proteins in dilute solutions in aqueous guanidine hydrochloride are analyzed to show that the data are not fit by either the random flight or wormlike chain models for linear chains. The analysis includes data on the intrinsic viscosity, root‐mean‐square radius of gyration, from small‐angle X‐ray scattering, and hydrodynamic radius, from the translational diffusion coefficient. It is concluded that residual structure consistent with that deduced from nuclear magnetic resonance on these solutions can explain the dilute solution results in a consistent manner through the presence of ring structures, which otherwise have an essentially flexible coil conformation. The ring structures could be in a state of continual flux and rearrangement. Calculation of the radius of gyration for the random‐flight model gives a similar reduction of this measure for chains joined at their endpoints, or those containing loop with two dangling ends, each one‐fourth the total length of the chain. This relative insensitivity to the details of the ring structure is taken to support the behavior observed across a range of proteins.     

Antimitotics induced cardiolipin cluster formation possible role in mitochondrial enzyme inactivation

We demonstrate here that drugs which inactivate cytochrome c oxidase are able to segregate cardiolipin essential for the enzyme activity, in a separate phase inaccessible for the enzyme. A molecular explanation of the drug-induced aggregation process is proposed.     

A particular hydrophobic cluster in the residual structure of reduced lysozyme drastically affects the amyloid fibrils formation

Six hydrophobic clusters involved in long-range interaction have been identified in the residual structure of reduced lysozyme at pH 2. Recently, it was found that modulation in the residual structure affected amyloid formation. In this paper, we examined the effect of the hydrophobic cluster containing W111 (cluster 5) on amyloid fibril formation of reduced lysozyme. The reduced W62G lysozyme, in which most of the hydrophobic clusters except for cluster 5 are disrupted, formed hardly any amyloid fibrils in comparison with the reduced wild-type. However, the disruption of cluster 5 by the mutation of Trp111 to Gly allowed significant amyloid fibril formation of reduced W62G lysozyme. Moreover, the extent of amyloid formation in the reduced W62G/W111G lysozyme was greater than that of the reduced wild-type lysozyme. From the above results, it became clear that cluster 5 contributed to retarding the amyloid fibrils formation of the W62G lysozyme.     

Shear-thickening and shear-induced pattern formation in starch solutions

Since Dintzis et al. reported shear-thickening behavior and shear-induced pattern formation in semidilute starch solutions for the first time in 1995, considerable efforts have been made to understand the science behind these observations. Despite these efforts, however, many questions regarding this behavior of starch solutions remain. Using a Brookfield programmable rheometer and a custom-built shear microscope, starch solutions in alkaline solution medium were investigated. In this report, we present data leading to the following conclusions: (1) gently prepared starch solutions are macroscopically heterogeneous with regions of highly concentrated gel-like structures dispersed in dilute starch solution; (2) shear breaks up these heterogeneous regions, increasing in viscosity (shear-thickening) which is thus seen to be a result of an increase in the concentration of dissolved starch; (3) pattern formation, observed when the solution is exposed to higher shear rate, is the result of a separate shear-induced aggregation process; and (4) aggregations are not induced below a certain critical threshold shear rate and time is also a factor in the behavior of the aggregate.     

Hydrophobic salts markedly diminish viscosity of concentrated protein solutions

Reducing viscosities of concentrated solutions of therapeutic proteins is important for their subcutaneous and intravenous delivery. Although inorganic salts and optimizing the pH were previously reported to dramatically lower the viscosity of a monoclonal antibody solution, herein we have determined these effects not to be general. Separately, we have found that hydrophobic ionic excipients, both anionic and cationic, substantially decrease the viscosity of concentrated (300–400 mg/mL) aqueous solutions of bovine serum albumin and γ‐globulin. The more hydrophobic the excipient, the greater its viscosity‐lowering effect is. With cationic ones, the concomitant contribution of the counter‐ion broadly follows the chaotropic order. The most potent excipients lower the viscosity over fourfold to levels far below the 50 cP threshold for subcutaneous injections. The observed viscosity reductions are rationalized in terms of three‐dimensional transient protein networks formed in concentrated solutions due to hydrophobic and, to a lesser extent, ionic interactions. These reversible protein aggregates are responsible for strong resistance to flow in concentrated protein solutions and hence their high viscosity; hydrophobic ions apparently effectively compete for these interprotein interactions, thereby giving rise to less viscous solutions. Biotechnol. Bioeng. 2011; 108:632–636. © 2010 Wiley Periodicals, Inc.     

A modular domain of NifU,a nitrogen fixation cluster protein,is highly conserved in evolution

HnifU, a gene exhibiting similarity tonifU genes of nitrogen fixation gene clusters, was identified in the course of expressed sequence tag (EST) generation from a human fetal heart cDNA library. Northern blot of human tissues and polymerase chain reaction (PCR) using human genomic DNA verified that the hnifU gene represented a human gene rather than a microbial contaminant of the cDNA library. Conceptual translation of the hnifU cDNA yielded a protein product bearing 77% and 70% amino acid identity to NifU-like hypothetical proteins fromHaemophilus influenzae andSaccharomyces cerevisiae, respectively, and 40–44% identity to the N-terminal regions of NifU proteins from several diazatrophs (i.e., nitrogen-fixing organisms). Pairwise determination of amino acid identities between the NifU-like proteins of nondiazatrophs showed that these NifU-like proteins exhibited higher sequence identity to each other (63–77%) than to the diazatrophic NifU proteins (40–48%). Further, the NifU-like proteins of non-nitrogenfixing organisms were similar only to the N-terminal region of diazatrophic NifU proteins and therefore identified a novel modular domain in these NifU proteins. These findings support the hypothesis that NifU is indeed a modular protein. The high degree of sequence similarity between NifU-like proteins from species as divergent as humans andH. influenzae suggests that these proteins perform some basic cellular function and may be among the most highly conserved proteins. Correspondence to: C.-C. Liew     

Cooperation between melanoma cell states promotes metastasis through heterotypic cluster formation

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Ethylene production,cluster root formation,and localization of iron(III) reducing capacity in Fe deficient squash roots

Dicots and non-graminaceous monocots have the ability to increase root iron(III) reducing capacity in response to iron (Fe) deficiency stress. In squash (Cucurbita pepo L.) seedlings, Fe(III) reducing capacity was quantified during early vegetative growth. When plants were grown in Fe-free solution, the Fe(III) reducing capacity was greatly elevated, reached peak activity on day 4, then declined through day 6. Root ethylene production exhibited a temporal pattern that closely matched that of Fe(III) reducing capacity through day 6. On the 7th day of Fe deficiency, cluster root morphology developed, which coincided with a sharp increase in the root Fe(III) reducing capacity, although ethylene production decreased. Localization of Fe(III) reducing capacity activity was observed during the onset of Fe deficiency and through the development of the root clusters. It was noted that localization shifted from an initial pattern which occurred along the main and primary lateral root axes, excluding the apex, to a final localization pattern in which the reductase appeared only on secondary laterals and cluster rootlets. This revised version was published online in June 2006 with corrections to the Cover Date.     

The failure of simple empirical relationships to predict the viscosity of mixed aqueous solutions of guanidine hydrochloride and glucose has important implications for the study of protein folding

Viscosities of aqueous solutions of guanidine hydrochloride (GuHCl) were measured in the presence of varying amounts of glucose. At high concentrations of glucose or GuHCl, the measured viscosities showed significant deviation from the values computed using a method proposed by Tanford (1966, J Biol Chem 241:3228-3232). This method was originally derived to allow the calculation of the effects of buffer or low concentrations of salts and other additives on the density and viscosity of aqueous solutions of urea or GuHCl. Recently it has been used to estimate the viscosity of denaturant solutions that contain high concentrations of viscogens. Our results show that the extrapolation of this approach to solutions of highly concentrated viscous co-solutes leads to significant errors. The implications for experimental studies of the viscosity dependence of conformational transitions in proteins is discussed.     

Ultra scale‐down of protein refold screening in microwells: Challenges,solutions and application

Steps for the refolding of proteins from solubilized inclusion bodies or misfolded product often represent bottlenecks in process development, where optimal conditions are typically derived empirically. To expedite refolding optimization, microwell screening may be used to test multiple conditions in parallel. Fast, accurate, and reproducible assays are required for such screening processes, and the results derived must be representative of the process at full scale. This article demonstrates the use of these microscale techniques to evaluate the effects of a number of additives on the refolding of IGF‐1 from denatured inclusion bodies, using an established HPLC assay for this protein. Prior to this, microwell refolding was calibrated for scale‐up using hen egg‐white lysozyme (HEWL) as an initial model protein, allowing us to implement and compare several assays for protein refolding, including turbidity, enzyme activity, and chromatographic methods, and assess their use for microwell‐based experimentation. The impact of various microplate types upon protein binding and loss is also assessed. Solution mixing is a key factor in protein refolding, therefore we have characterized the effects of different methods of mixing in microwells in terms of their impact on protein refolding. Our results confirm the applicability and scalability of microwell screening for the development of protein refolding processes, and its potential for application to new inclusion body‐derived protein products. Biotechnol. Bioeng. 2009;103: 329–340. © 2008 Wiley Periodicals, Inc.     

Diffusivity of CH4 In Model Silica Nanopores: Molecular Dynamics and Quasichemical Mean Field Theory

Equilibrium molecular dynamics and dual control volume grand canonical molecular dynamics experiments were carried out aiming at the investigation of the dependence of transport diffusivity upon the adsorbent pore size and sorbate concentration of CH₄ in cylindrical silica nanopores at 298?K, calibrated with respect to experimental data of zeolite VPI 5; the results of simulation were elaborated on the basis of the quasichemical mean field approximation via a theoretical model for surface diffusion. Our mapping procedure between simulation and quasichemical theory reveals that sorbate–sorbate energetics emerge as the physical reason for the variation of corrected (Darken) and hence transport diffusivity with respect to pore size and sorbed phase fractional occupancy.     

Habitat geometry, population viscosity and the rate of genetic drift

In all natural populations, individuals located close to one another tend to interact more than those further apart. The extent of population viscosity can have important implications for ecological and evolutionary processes. Here we develop a spatially explicit population model to examine how the rate of genetic drift depends upon both spatial population structure and habitat geometry. The results show that the time to fixation for a new and selectively neutral mutation is dramatically increased in viscous populations. Furthermore, in viscous populations the time to fixation depends critically on habitat geometry. Fixation time for populations of identical size increases markedly as landscape width decreases and length increases. We suggest that similar effects will also be important in metapopulations, with the spatial arrangement of subpopulations and their connectivity likely to determine the rate of drift. We argue that the recent increases in computer power should facilitate major advances in our understanding of evolutionary landscape ecology over the next few years, and suggest that the time is ripe for a unification of spatial population dynamics theory, landscape ecology and population genetics.